Design of a multiwalled carbon nanotube–Nafion–cysteamine modified tyrosinase biosensor and its adaptation of dopamine determination

In this work, a multiwalled carbon nanotube (MWCNT)–Nafion–cysteamine (CA) modified tyrosinase biosensor brings a new and original perspective to biosensor technology intended for the development of dopamine determination. Dopamine measurements were done at 0.2 V with the amperometric method by the developed biosensor system. In addition, in this study dopamine determination was carried out by using the differential pulse voltammetry method between potentials of 0.4 and −0.15 V. In the optimization studies of the biosensor, some parameters such as optimal pH, optimal temperature, optimal enzyme amount, and effect of MWCNT concentration were investigated. Afterward, in the characterization studies, some parameters such as linearity and reproducibility were determined. In the reproducibility experiment, an average value of 1.026 μM, a standard deviation of ±0.03975, and a coefficient of variation of 3.8% were determined for a 1-μM dopamine concentration (n = 15). Determination of dopamine was carried out in drug samples by the developed biosensor.     

Cryopreservation of turkey semen: Effect of breeding line and freezing method on post-thaw sperm quality,fertilization, and hatching

Cryopreservation methods for poultry semen are not reliable for germplasm preservation, especially for turkeys, where fertility rates from frozen/thawed semen are particularly low. The objective was to evaluate cryopreservation methods for effectiveness in promoting cryosurvival and post-thaw function of sperm from five turkey lines: one commercial line and four research (RBC1; E; RBC2; F) lines from Ohio State University (OSU). The model for cryopreservation was set up as a 2 × 2 × 2 × 5 design for cryoprotectant (glycerol or dimethylacetamide (DMA)), cryopreservation medium (Lake or ASG), method of dilution (fixed dilution volume versus fixed sperm concentration) and turkey line, respectively. The final cryoprotectant concentrations were 11% glycerol or 6% DMA. Thawed sperm were evaluated for plasma membrane integrity and quality, motility, acrosome integrity and, after artificial insemination, for egg fertility and hatchability. Commercial turkey hens were used for all fertility trials, regardless of semen source. Turkey sperm frozen with glycerol exhibited higher membrane integrity and membrane quality upon thawing than turkey sperm frozen with DMA although no differences in total motility, and only minimal differences in progressive motility, were detected among the eight cryopreservation treatments. Within line, fertility was affected by cryoprotectant, medium and dilution method, where the overall highest percentages of fertile, viable embryos (Day 7) occurred for the DMA/ASG/fixed sperm concentration method, while high percentages (15.8–31.5%) of fertile, non-viable embryos (Day 1–6) were observed for multiple cryopreservation methods, including two glycerol treatments. From a single insemination, the duration of true and viable fertility in all lines was 10–13 weeks and 9–10 weeks, respectively. The duration of hatchability was 4–6 weeks after insemination for four of the turkey lines. The highest percentage of viable embryos was observed for the commercial line (9.5 ± 2.4%), followed by the E line (5.3 ± 1.3%), F line (3.7 ± 2.0%) and RBC2 line (2.6 ± 0.8%). For the RBC1 line, there was 100% embryonic death by Day 6 of incubation. Overall, better fertility results were obtained with the cryoprotectant DMA, the ASG diluent and fixed sperm concentration. However, the applicability of this method for preserving semen from research populations may be line dependent.     

A liquid chromatography method for quantifying caffeine dissolution from pharmaceutical formulations into colloidal,fat-rich media

A simple and rapid high-performance liquid-chromatography method is presented that permits quantification of caffeine in colloidal fat emulsions proposed as new ‘biorelevant’ dissolution media (Intralipid™ and various milks). Using a mobile phase of 0.1 M sodium acetate (pH 4.0) and acetonitrile (89.5:10.5, v/v) at 1 ml min⁻¹, the drug and internal standard (7-β-hydroxyethyltheophylline) were eluted within 8 min. Caffeine extraction was undertaken by protein precipitation in ice-cold 12% (w/v) trichloroacetic acid and centrifugation at 10,000 rpm for 15 min. This simple extraction method generated caffeine recovery values (corrected for % fat content) of 75.4 ± 1.4–100.6 ± 5.5%. The limit of detection was within the range 0.25–0.4 μg ml⁻¹ and linearity was demonstrated in each medium up to 125 μg ml⁻¹. Precision was <11.5% RSD and intra- and inter-day accuracy was 93.4–109.3%. The validated method was applied to in vitro USP dissolution tests in milk which compared the kinetics of caffeine release from (i) extended release matrices containing hydroxypropyl methylcellulose (HPMC) and (ii) an immediate release commercial analgesic tablet. Good reproducibility was obtained in both extended and immediate release dissolution tests. The method provides high-throughput quantification of this common drug in fat emulsions used as biorelevant dissolution media.     

Discrepancy between radioimmunoassay and high performance liquid chromatography tandem-mass spectrometry for the analysis of androstenedione

The discrepancy of results for the quantification of androstenedione in human serum between a radioimmunoassay (RIA) method and high performance liquid chromatography tandem-mass spectrometry (LC–MS/MS) was investigated. RIA overestimated concentrations compared to LC–MS/MS on 59 clinical samples (RIA = 1.79 × LC–MS/MS + 0.94). RIA kit and LC–MS/MS calibrants were also determined by both methods. The RIA performed with improved accuracy on the calibrants (RIA = 1.35 × LC–MS/MS − 0.28). Lipid, protein, electrolyte content, and pH of the two sets of calibrants were further investigated. The RIA calibrants contained little lipid material, while the LC–MS/MS calibrant material contained the same levels expected in normal serum/plasma. The pH and sex hormone binding globulin (SHBG) values were different between the RIA calibrants and the LC–MS/MS calibrant material (SHBG, 31 ± 2 and 38 ± 2 nmol/l; pH, 8.27 ± 0.18 and 8.66 ± 0.03, respectively). No correlation was observed between androstenedione RIA and LC–MS/MS discrepancy and lipid or protein. LC–MS/MS sample preparation was tested for the removal of protein-bound material and recovery determined (99–108%). The corresponding RIA results overestimated androstenedione by 52–174% compared to LC–MS/MS. The results here demonstrate that LC–MS/MS is the more accurate method.     

Lambing rates and litter size following carazolol administration prior to insemination in Kivircik ewes

The effect of carazolol on the ease of penetrating the cervix during artificial insemination, lambing rate and litter size was studied using 1.5–4.0-year old Kivircik ewes in an incomplete 3 × 2 × 2 experimental design. All of the ewes in this study were synchronized for oestrus by insertion of a progesterone impregnated vaginal sponge for 12 days and administration of 400 IU PMSG at sponge withdrawal. Three methods of service were compared: natural service, artificial insemination (AI) with fresh semen, or AI with frozen semen. Two times of insemination (fixed time AI versus AI at observed oestrus) were compared on the fresh and frozen AI treatments. The absence (control) or use of carazolol (carazolol; 0.5 mg/ewe i.m. 30 min before mating) was the third factor in the design and penetration of the cervix by the insemination pipette was assessed as shallow (<10 mm), middle (10–20 mm) or deep (>20 mm). Natural service ewes were only mated at observed oestrus. Consequently, the factorial design was incomplete and there were a total of 10 treatments each represented by 30 ewes. Natural service resulted in a significantly (P < 0.05) higher lambing rate and litter size (86%; 2.0 ± 0.05 lambs/ewe) than AI using fresh (65%; 1.6 ± 0.1 lambs/ewe) or frozen (40%; 1.4 ± 0.14 lambs/ewe) semen. For AI animals the lambing rate and litter size were not significantly different when service was at a fixed time (50%; 1.5 ± 0.12 lambs/ewe) or at observed oestrus (56%; 1.5 ± 0.12 lambs/ewe). Carazolol did not permit complete cervical penetration in any ewe. Deep penetration of the cervix at AI was achieved in 33% of untreated (control) and 48% of carazolol treated ewes (P < 0.05). However, the proportion of ewes in which penetration of the cervix and semen deposition was greater than shallow was similar for control (82%) and carazolol (85%), and lambing rate and litter size were similar for both treatments. Over the three service methods, the lambing rate was 56% for control and 63% for carazolol (NS) and litter size was similar for both treatments. It was concluded that the carazolol treatment used prior to natural mating or AI in this experiment did not improve lambing rate or litter size in Kivircik ewes.     

Male reproductive traits, semen cryopreservation, and heterologous in vitro fertilization in the bobcat (Lynx rufus)

There is limited information on bobcat ejaculate traits and sperm cryopreservation and fertilizing ability. Bobcats were electroejaculated under general anesthesia in November (autumn) and April (spring), and endocrine and sperm traits were characterized. Testosterone (mean ± SEM: 0.90 ± 0.15 ng/mL) was not different between sampling times, but cortisol (average: 13.95 ± 1.73 μg/dL) was significantly higher in April. Average number of spermatozoa was 10.0 ± 3.4 × 10⁶ sperm/ejaculate, with values being significantly higher in April. Sperm motility (average 55.7 ± 5.8% motile sperm) was not different between sampling times. The proportion of normal spermatozoa in the ejaculate (average: 14.7 ± 2.1%) was significantly higher in April, but the percentage of spermatozoa with intact acrosomes (average: 43.7 ± 3.8%) was significantly higher in autumn. Spermatozoa were cryopreserved in a Tes-Tris-based diluent (TEST) or Biladyl, both containing 20% egg yolk and 4% glycerol. Diluted sperm were loaded into straws, refrigerated using a programmable thermoblock with a dry chamber, frozen in nitrogen vapors, thawed, and incubated in F-10 medium with 5% fetal bovine serum for up to 3 h. After cryopreservation in TEST, there were about 50% motile sperm upon thawing, and survival was high during incubation post-thaw. Cryopreservation in Biladyl led to similar results, but motility decreased substantially during incubation post-thaw. Bobcat spermatozoa fertilized domestic cat oocytes matured in vitro. Fertilization rates were higher for sperm collected in April and cryopreserved in TEST (46%) than for those cryopreserved using Biladyl (<3%). Fertilized oocytes cleaved in culture, and some (27%) reached the morula stage. This study has allowed us to gain further baseline information on bobcat reproduction, explore sperm cryopreservation conditions, and show that fertilizing capacity can be tested using in vitro-matured cat oocytes. These results will be important for future conservation efforts.     

The effect of insemination time and sperm dose on pregnancy rate using sex-sorted ram sperm

The objective was to determine the optimum timing of insemination and minimum effective dose rate of sex-sorted ram sperm. Semen from three Merino rams was sorted into high purity X- and Y-chromosome bearing sperm populations. Ovulation was controlled in 732 Merino ewes using PMSG at progestagen pessary removal and GnRH 36 h later. Sorted (S) and non-sorted (NS) doses of 1 or 15 × 10⁶ motile, frozen-thawed sperm were inseminated laparoscopically at 50, 54, 58, 62, and 66 h after progestagen withdrawal. An additional treatment dose of 0.5 × 10⁶ S or NS sperm was inseminated at the 58 h time point (n = 60). Pregnancy was diagnosed by ultrasound at 60-62 d gestation. Both 1 × 10⁶ and 15 × 10⁶ sperm achieved similar pregnancy rates, regardless of sperm type, at 58 h (S1: 46 ± 9.4%; S15: 43 ± 9.3%; NS1: 41 ± 9.2%; NS15: 49 ± 9.4%). However, pregnancy rates were lower (P < 0.05) for doses of 1 than 15 × 10⁶ sperm inseminated at 50 (15 ± 6.3% vs. 36 ± 9.1%), 54 (14 ± 4.4% vs. 55 ± 7.3%), 62 (33 ± 6.9% vs. 54 ± 7.3%), and 66 h (29 ± 8.6% vs. 56 ± 9.5%). There was no difference between S and NS sperm for inseminations with 0.5 × 10⁶ motile sperm at 58 h after PR (15 ± 3.6% vs. 14 ± 3.3%), nor with 15 × 10⁶ motile sperm at all insemination times (49 ± 6.3% vs. 49 ± 6.3%). However, fertility was higher for S than NS sperm at the 1 × 10⁶ dose level (37 ± 6.1% and 16 ± 4.0%). More than 90% of lambs born were of the predicted sex. We hypothesise that the sorting process selects a homogeneous, fertile sub-population of sperm, removing those that are dead, damaged and morphologically abnormal.     

Capillary electrophoresis with noncovalently bilayer-coated capillaries for stability study of allergenic proteins in simulated gastrointestinal fluids

A novel noncovalently bilayer-coated capillary using cationic polymer polybrene (PB) and anionic polymer (sodium 4-styrenesulfonate) (PSS) as coatings was prepared. This PB–PSS coating showed good migration-time reproducibility for proteins and high stability in the range of pH 2–10 and in the presence of 1 M NaOH, acetonitrile and methanol. Capillary electrophoresis with PB–PSS coated capillaries was successfully applied to quantitatively investigate the stability of bovine serum albumin, ovomucoid, β-lactoglobulin and lysozyme in simulated gastrointestinal fluids. β-lactoglobulin A and β-lactoglobulin B were both stable in simulated gastric fluid with degradation percentages of 34.3% and 17.2% after 60 min of incubation, respectively. Bovine serum albumin, ovomucoid and lysozyme were stable in simulated intestinal fluid with degradation percentages of 17.7%, 23.4% and 22.8% after 60 min of incubation, respectively. The superiority of the proposed method over sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and capillary electrophoresis with untreated fused silica capillaries was demonstrated and emphasized.     

Binding of bivalent ions to actinomycete mannanase is accompanied by conformational change and is a key factor in its thermal stability

The study aimed to define the key factors involved in the modulation of actinomycete mannanases. We focused on the roles of carbohydrate-binding modules (CBMs) and bivalent ions. To investigate the effects of these factors, two actinomycete mannanase genes were cloned from Streptomyces thermoluteus (StManII) and Streptomyces lividans (SlMan). CBMs fused to mannanase catalytic domains do not affect the thermal stability of the proteins. CBM2 of StManII increased the catalytic efficiency toward soluble-mannan and insoluble-mannan by 25%–36%, and CBM10 of SlMan increased the catalytic efficiency toward soluble-mannan by 40%–50%. Thermal stability of wild-type and mutant enzymes was enhanced by calcium and manganese. Thermal stability of SlMandC was also slightly enhanced by magnesium. These results indicated that bivalent ion-binding site responsible for thermal stability was in the catalytic domains. Thermal stability of mannanase differed in the kinds of bivalent ions. Isothermal titration calorimetry revealed that the catalytic domain of StManII bound bivalent ions with a K_a of 5.39 ± 0.45 × 10³–7.56 ± 1.47 × 10³ M^− 1, and the catalytic domain of SlMan bound bivalent ions with a K_a of 1.06 ± 0.34 × 10³–3.86 ± 0.94 × 10³ M^− 1. The stoichiometry of these bindings was consistent with one bivalent ion-binding site per molecule of enzyme. Circular dichroism spectrum revealed that the presence of bivalent ions induced changes in the secondary structures of the enzymes. The binding of certain bivalent ion responsible for thermal stability was accompanied by a different conformational change by each bivalent ion. Actinomycete mannanases belong to GHF5 which contained various hemicellulases; therefore, the information obtained from mannanases applies to the other enzymes.     

Cryopreservation of sperm of an indigenous endangered fish species Nandus nandus (Hamilton, 1822) for ex-situ conservation

This study dealt with the development of cryopreservation protocol for Nandus nandus, which entailed a number of experiments. Sperm was collected by sacrificing males. The collected sperm was suspended in extenders. Activation of sperm motility was evaluated in different osmolalities of NaCl. Motility of sperm decreased as the osmolality of the extender increased and was completely inhibited at almost 319 mOsmol/kg. To evaluate the toxicity of cryoprotectant, sperm was incubated with DMSO, methanol and ethanol at 5%, 10% and 15% concentrations, respectively, for 5–35 min. Five and ten percent of cryoprotectants produced better motility during 5 and 10 min incubation. Sperm incubated with 15% cryoprotectant seemed to be toxic and this concentration was excluded in the subsequent trials. Three extenders, namely, Alsever’s solution, egg-yolk citrate and urea egg-yolk and three cryoprotectants, DMSO, methanol and ethanol were employed to preserve the sperm. Alsever’s solution with 10% DMSO showed best performance producing 90.0 ± 1.8% and 75.0 ± 2.5% equilibration and post-thaw motility followed by that of 82.5 ± 4.2% and 62.5 ± 5.5% with Alsever’s solution plus methanol, respectively. Between two diluents, sperm preserved with Alsever’s solution plus DMSO produced highest fertilization (76.7 ± 3.3%) and hatching (43.8 ± 7.9%) while fresh sperm yielded 83.3 ± 6.7% and 64.0 ± 10.4% fertilization and hatching, respectively. The protocol developed through the study can be applied for long-term conservation of genetic materials of the endangered fish N. nandus and the cryopreserved sperm can be used in artificial breeding for generating new individuals.     

Interactions of KChIP4a and its mutants with Ca or Kv4.3 N-terminus by affinity capillary electrophoresis

The specific binding of auxiliary Kv channel-interacting proteins (KChIPs) to the N terminus of Kv4 pore-forming α-subunits results in modulation of gating properties, surface expression, and subunit assembly of Kv4 channels. However, the interactions between KChIPs and Kv4 remain elusive. Thus, affinity capillary electrophoresis (ACE) was employed to quantitatively evaluate the interactions between KChIPs and Kv4.3 N terminus (KvN) and between KChIP4a/related mutants and Ca²⁺ for the first time. The mobility ratio, derivatives calculated from the mobility shift method, was used to deduce the binding constants (K_b). As a result, the binding constants for KChIP4a/KvN and KChIP1/KvN complexes were (8.32 ± 1.66) × 10⁶ L mol^–1 and (5.26 ± 0.71) × 10⁶ L mol^–1, respectively. In addition, in the presence of calcium (10 μmol L^–1), the binding constant of KChIP4a/KvN increased to (6.72 ± 1.66) × 10⁷ L mol^–1. In addition, the binding constant of KChIP4a with Ca²⁺ was (7.1 ± 1.5) × 10⁷ L mol^–1. Besides, studies on the effect of truncated mutants revealed that the third EF hand of KChIP4a was related to high-affinity binding with Ca²⁺, and the integrity of the molecular structure of KChIP4a was important for Ca²⁺ binding. This method profits from small samples, rapid analysis, and simple operation without being time-consuming.     

Determination of human serum 8-hydroxy-2′-deoxyguanosine (8-OHdG) by HPLC-ECD combined with solid phase extraction (SPE)

Human serum 8-hydroxy-2′-deoxyguanosine (8-OHdG) was measured by HPLC-ECD method combined with solid phase extraction (SPE) developed by our group: (our proprietary kit, named 8-OHdG Pre-treatment Kit (TANITA Corporation)). The major interfering substances and proteins in serum were removed by 8-OHdG Pre-treatment Kit. This measurement method was highly reproducible (CV = 2.2–7.1%) and demonstrated the lower detection limit for control serum sample of less than 10 pg/ml without the sample evaporation. The other hand 8-OHdG concentration in serum for healthy people was in the range of 0–70 pg/ml (25.5 ± 13.8 pg/ml, n = 37). Secondary a relationship between the HPLC-ECD and ELISA methods was investigated. ELISA method could not detect 8-OHdG concentration in serum for healthy people, because the detection limit of 130 pg/ml was higher than the normal range for healthy people. These results show our SPE method has high sensitivity and quantitative accuracy for 8-OHdG analysis.     

Determination and pharmacokinetics of gastrodin in human plasma by HPLC coupled with photodiode array detector

In present study, an HPLC method coupled with photodiode array detector (HPLC-PDA) was established for determination and pharmacokinetics of gastrodin (GAS) in human plasma after an oral administration of GAS capsule. In the method, ethanol and dichloromethane were respectively used for deproteinization and purification during the sample preparation procedure. Separation of GAS was achieved on an AichromBond-AQ C18 column (5 μm, 150 mm × 4.6 mm) with the mobile phase of methanol–0.1% phosphoric acid solution (2:98, v/v) at a flow rate of 0.8 ml/min. The wavelength was set at 220 nm and the injection volume was 20 μl. Under the conditions, the calibration curve was linear within the concentration range of 50–4000 ng/ml with the correlation coefficient (r) of 0.99554 (weight = 1/X²) and the lower limit of quantification (LLOQ) was 50 ng/ml. The inter- and intra-day precisions were less than 11% and the accuracies (%) were within the range of 95.55–103.78%. The extraction recoveries were over 65% with RSDs less than 5.50%. The GAS was proved to be stable under tested conditions. Thus, the method was valid enough to be applied for pharmacokinetic study of GAS in human plasma. The pharmacokinetic parameters of GAS in human plasma after an oral administration of 200 mg GAS capsule were described as: C_max, 1484.55 ± 285.05 ng/ml; T_max, 0.81 ± 0.16 h; t_1/2α, 3.78 ± 2.33 h; t_1/2β, 6.06 ± 3.20 h; t_1/2Ka, 0.18 ± 0.53 h; K₁₂, 0.18 ± 0.41/h; K₂₁, 0.20 ± 0.16/h; K₁₀, 4.11 ± 15.81/h; V1/F, 180.35 ± 89.44 L; CL/F, 62.50 ± 140.03 l/h; AUC_0→t, 5619.41 ± 1972.88 (ng/ml) h; and AUC_0→∞, 7210.26 ± 3472.74 (ng/ml) h, respectively. These will be useful for the clinical application of GAS.     

Epigenetic modulation of RFC1, MHC2TA and HLA-DR in systemic lupus erythematosus: Association with serological markers and six functional polymorphisms of one-carbon metabolic pathway

The current study was conducted to elucidate the effect of genetic variations in one-carbon metabolism on the epigenetic regulation of major histocompatibility complex II transactivator (MHC2TA), reduced folate carrier 1 (RFC1/SLC19A1) and human leukocyte antigen (HLA)-DR in systemic lupus erythematosus (SLE). PCR-RFLP/AFLP, bisulfite-sequencing and real-time PCR approaches were used for genetic, epigenetic and expression analysis respectively. SLE cases exhibited elevated plasma homocysteine levels compared to healthy controls (24.93 ± 1.3 vs. 11.67 ± 0.48 μmol/l), while plasma folate levels showed no association (7.10 ± 2.49 vs. 7.64 ± 2.09 ng/ml). The RFC1 80G>A polymorphism showed 1.32-fold risk (95% CI: 1.02–1.72) for SLE, while glutamate carboxypeptidase II (GCPII) 1561C>T showed reduced risk (OR: 0.47, 95% CI: 0.24–0.90). The expression of RFC1 (0.37 ± 0.09 vs. 0.60 ± 0.17) and HLA-DR (0.68 ± 0.17 vs. 0.98 ± 0.02) was down regulated in the SLE cases. The hypermethylation of RFC1 as observed in the current study may contribute for its down regulation. Plasma folate and thymidylate synthase (TYMS) 5′-UTR 28 bp tandem repeat showed an inverse association with methylation of RFC1 and MHC2TA. SLE cases with hypocomplementemia showed hypermethylation of RFC1, hypomethylation/up regulation of MHC2TA and down regulation of HLA-DR. The hypermethylation of MHC2TA and down regulation of RFC1, MHC2TA and HLA-DR were observed in anti-cardiolipin antibody positive SLE cases. The up regulation of RFC1 and HLA-DR was observed in anti-dsDNA antibody positive SLE cases. The hypomethylation/upregulation of RFC1 and MHC2TA was observed in anti-RNP antibody positive cases. To conclude, one-carbon genetic variants influence epigenetic of MHC2TA and RFC1, thus contributing to phenotypic heterogeneity of SLE.     

Studies on the regioselectivity and kinetics of the action of trypsin on proinsulin and its derivatives using mass spectrometry

Human M-proinsulin was cleaved by trypsin at the R³¹R³²–E³³ and K⁶⁴R⁶⁵–G⁶⁶ bonds (B/C and C/A junctions), showing the same cleavage specificity as exhibited by prohormone convertases 1 and 2 respectively. Buffalo/bovine M-proinsulin was also cleaved by trypsin at the K⁵⁹R⁶⁰–G⁶¹ bond but at the B/C junction cleavage occurred at the R³¹R³²–E³³ as well as the R³¹–R³²E³³ bond. Thus, the human isoform in the native state, with a 31 residue connecting C-peptide, seems to have a unique structure around the B/C and C/A junctions and cleavage at these sites is predominantly governed by the structure of the proinsulin itself. In the case of both the proinsulin species the cleavage at the B/C junction was preferred (65%) over that at the C/A junction (35%) supporting the earlier suggestion of the presence of some form of secondary structure at the C/A junction. Proinsulin and its derivatives, as natural substrates for trypsin, were used and mass spectrometric analysis showed that the k_cat./K_m values for the cleavage were most favourable for the scission of the bonds at the two junctions (1.02 ± 0.08 × 10⁵ s^− 1 M^− 1) and the cleavage of the K²⁹–T³⁰ bond of M-insulin-RR (1.3 ± 0.07 × 10⁵ s^− 1 M^− 1). However, the K²⁹–T³⁰ bond in M-insulin, insulin as well as M-proinsulin was shielded from attack by trypsin (k_cat./K_m values around 1000 s^− 1 M^− 1). Hence, as the biosynthetic path follows the sequence; proinsulin → insulin-RR → insulin, the K²⁹–T³⁰ bond becomes shielded, exposed then shielded again respectively.     

Development of high performance liquid chromatography/electrospray ionization mass spectrometry for assay of ginkgolic acid (15:1) in rat plasma and its application to pharmacokinetics study

A highly sensitive HPLC–ESI-MS method has been developed and validated for the quantification of ginkgolic acid (15:1) in a small quantity of rat plasma (50 μL) using its homologous compound ginkgolic acid (17:1) as an internal standard. GA (15:1) and GA (17:1) were extracted from biological matrix by direct protein precipitation with 5-fold volume of methanol and separated on an Elite hypersil BDS C₁₈ column (2.1 × 100 mm, 3 μm), eluted with acetonitrile:water (92:8, v/v, containing 0.3% glacial acetic acid). Linear range was 8–1000 ng/mL with the square regression coefficient (r²) of 0.996. The lowest concentration (8 ng/mL) in the calibration curve was estimated as LLOQ with both deviation of accuracy and RSD of precision <20% (n = 6). The intra- and inter-day precision ranged from 3.6% to 9.9%, and the intra- and inter-day accuracy was between 89.9% and 101.3%. This method was successfully applied to study pharmacokinetics of GA (15:1) in rats after oral administration at a dose of 10 mg/kg. GA (15:1) pharmacokinetic parameters C_max, T_max, t_1/2, AUC_0–12h are 1552.9 ± 241.0 ng/mL, 0.9 ± 0.7 h, 5.5 ± 2.6 h, 3356.0 ± 795.3 ng h/mL, respectively.     

In vitro characteristics of frozen-thawed, sex-sorted bull sperm after refreezing or incubation at 15 or 37 °C

The objective was to determine the in vitro characteristics of frozen-thawed dairy bull sperm after sex-sorting and refreezing and thawing (0, 2, and 4 h post-thaw; 37 °C) or post-sort incubation at 15 or 37 °C for 30 and 24 h, respectively. These sperm were compared with nonsorted frozen-thawed sperm (control) and with nonsorted sperm undergoing two cryopreservation procedures (FF; 0, 2, and 4 h). Frozen-thawed sex-sorted (FS) sperm maintained at 15 or 37 °C had higher (P < 0.001) progressive motility (PM), velocity, mitochondrial function, viability, and acrosome integrity than that of control sperm but similar total motility at 0 and 2 h of incubation. Frozen-thawed sex-sorted sperm incubated at 15 °C maintained high levels of motility (66.5 ± 1.6%) and viability/acrosome integrity (64.9 ± 1.2%) at 24 h incubation and, after rewarming and further 6 h incubation at 37 °C, acceptable levels of motility (35.8 ± 1.6%) and viability/acrosome integrity (51.2 ± 1.2%) were maintained. Frozen-thawed sex-sorted sperm maintained at 37 °C had lower levels of motility, integrity, mitochondrial respiration, and velocity from 4 h of incubation onward than that of those incubated at 15 °C. However, when frozen-thawed sex-sorted sperm were refrozen (FSF), motility and velocity were depressed at all hours compared with levels exhibited by control sperm, but membrane viability/acrosome integrity and mitochondrial respiration were similar at 0 and 2 h post-thaw. Acrosome integrity of sperm in all groups undergoing sorting was exceptionally high at 0 h (≥90%), even after re-cryopreservation and 4 h of incubation (77.5 ± 1.3%). Double frozen-thawed nonsorted sperm (FF) had similar motility to FSF sperm at 0 and 2 h post-thaw but at all time points had the lowest (P < 0.001) levels of acrosome intact/viable sperm and mitochondrial respiration and the lowest velocity at 0 h. In conclusion, whereas sex-sorting improved the functionality of frozen-thawed sperm, refreezing depressed motility, viability, and velocity but not acrosome integrity after extended incubation compared with that of control sperm. Furthermore, frozen-thawed, sex-sorted sperm may be stored for transport at 15 °C for up 24 h without detrimental effects on in vitro sperm characteristics.     

Responses of a fringing Cyperus papyrus L. swamp to changes in water level

Over a 9-year period (1993–2001), the land-water width of a papyrus fringe on the southern shore of Lake Naivasha, Kenya, varied between 40 and 60 m. Increases in width via rhizome spreading into open water followed the 1997/1998 El Niño flood when water depths rose by about 2 m. Germination of papyrus seeds also responded to water depth with a mean ± S.E. rate in experiments of 23 ± 6% after 21 days when water level was 5 cm below the sediment surface. No germination occurred when sediment was flooded or allowed to desiccate. Rhizome spreading from floating mats appeared to be favoured by deep water with seedling spread favoured on newly inundated, low-gradient slopes in shallow water. Although natural regenerative capacity was influenced by water depth, the height, density, biomass and chemical content of papyrus were not. Total average biomass along a land-water transect was 6950 ± 860 g m⁻² which was large in relation to nutrient and mineral contents. Culms contained 0.47 ± 0.14% N and 0.06 ± 0.05% P and rhizomes 0.71 ± 0.21% and 0.10 ± 0.06%. Sediment underlying the swamp was aerobic and there were small land-water gradients in the BOD of swamp water and sediment. However, chemical gradients were weak compared with wider papyrus swamps elsewhere. Lake and swamp water mixed in the narrow fringe studied and residence times for organic matter may not have been long enough for organic material to mineralise before entering lake water.     

Confirmatory analysis of buprenorphine,norbuprenorphine, and glucuronide metabolites in plasma by LCMSMS. Application to umbilical cord plasma from buprenorphine-maintained pregnant women

An LCMSMS method was developed and fully validated for the simultaneous quantification of buprenorphine (BUP), norbuprenorphine (NBUP), buprenorphine-glucuronide (BUP-Gluc), and norbuprenorphine-glucuronide (NBUP-Gluc) in 0.5 mL plasma, fulfilling confirmation criteria with two transitions for each compound with acceptable relative ion intensities. Transitions monitored were 468.3 > 396.2 and 468.3 > 414.3 for BUP, 414.3 > 340.1 and 414.3 > 326.0 for NBUP, 644.3 > 468.1 and 644.3 > 396.3 for BUP-Gluc, and 590.3 > 414.3 and 590.3 > 396.2 for NBUP-Gluc. Linearity was 0.1–50 ng/mL for BUP and BUP-Gluc, and 0.5–50 ng/mL for NBUP and NBUP-Gluc. Intra-day, inter-day, and total assay imprecision (%RSD) were <16.8%, and analytical recoveries were 88.6–108.7%. Extraction efficiencies ranged from 71.1 to 87.1%, and process efficiencies 48.7 to 127.7%. All compounds showed ion enhancement, except BUP-Gluc that demonstrated ion suppression: variation between 10 different blank plasma specimens was <9.1%. In six umbilical cord plasma specimens from opioid-dependent pregnant women receiving 14–24 mg/day BUP, NBUP-Gluc was the predominant metabolite (29.8 ± 7.6 ng/mL), with BUP-Gluc (4.6 ± 4.8 ng/mL), NBUP (1.5 ± 0.8 ng/mL) and BUP (0.4 ± 0.2 ng/mL). Although BUP biomarkers can be quantified in umbilical cord plasma in low ng/mL concentrations, the significance of these data as predictors of neonatal outcomes is currently unknown.